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Asset Tag: PacBio RS II

September 22, 2019

Investigating the central metabolism of Clostridium thermosuccinogenes.

Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in Escherichia coli and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.IMPORTANCE Running industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production. Clostridium thermosuccinogenes seems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PPi cofactor utilization. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Redefinition and unification of the SXT/R391 family of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in Vibrio cholerae, the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5′ end of the chromosomal gene prfC Hence, the integrase gene int has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in GenBank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in V. cholerae isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh-pandemic toxigenic V. cholerae strains O1 and O139. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps, suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together, these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family.IMPORTANCEVibrio cholerae is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh-pandemic V. cholerae but also in several species of Enterobacteriaceae Most epidemiological surveys use the integrase gene as a marker to screen for SXT/R391 ICEs in clinical or environmental strains. With the recent reports of closely related elements that carry an alternative integrase gene, it became urgent to investigate whether ICEs that have been left out of the family are a liability for the accuracy of such screenings. In this study, based on comparative genomics, we broaden the SXT/R391 family of ICEs to include atypical ICEs that are often associated with heavy metal resistance. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.

Lactobacillus plantarum (L. plantarum) K25 is a probiotic strain isolated from Tibetan kefir. Previous studies showed that this exopolysaccharide (EPS)-producing strain was antimicrobial active and cold tolerant. These functional traits were evidenced by complete genome sequencing of strain K25 with a circular 3,175,846-bp chromosome and six circular plasmids, encoding 3365 CDSs, 16 rRNA genes and 70 tRNA genes. Genomic analysis of L. plantarum K25 illustrates that this strain contains the previous reported mechanisms of probiotic functionality and cold tolerance, involving plantaricins, lysozyme, bile salt hydrolase, chaperone proteins, osmoprotectant, oxidoreductase, EPSs and terpenes. Interestingly, strain K25 harbors more genes that function in defense mechanisms, and lipid transport and metabolism, in comparison with other L. plantarum strains reported. The present study demonstrates the comprehensive analysis of genes related to probiotic functionalities of an EPS-producing L. plantarum strain based on whole genome sequencing.

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September 22, 2019

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.© 2018 Zhu et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Genome biology of a novel lineage of planctomycetes widespread in anoxic aquatic environments.

Anaerobic strains affiliated with a novel order-level lineage of the Phycisphaerae class were retrieved from the suboxic zone of a hypersaline cyanobacterial mat and anoxic sediments of solar salterns. Genome sequences of five isolates were obtained and compared with metagenome-assembled genomes representing related uncultured bacteria from various anoxic aquatic environments. Gene content surveys suggest a strictly fermentative saccharolytic metabolism for members of this lineage, which could be confirmed by the phenotypic characterization of isolates. Genetic analyses indicate that the retrieved isolates do not have a canonical origin of DNA replication, but initiate chromosome replication at alternative sites possibly leading to an accelerated evolution. Further potential factors driving evolution and speciation within this clade include genome reduction by metabolic specialization and rearrangements of the genome by mobile genetic elements, which have a high prevalence in strains from hypersaline sediments and mats. Based on genetic and phenotypic data a distinct group of strictly anaerobic heterotrophic planctomycetes within the Phycisphaerae class could be assigned to a novel order that is represented by the proposed genus Sedimentisphaera gen. nov. comprising two novel species, S. salicampi gen. nov., sp. nov. and S. cyanobacteriorum gen. nov., sp. nov.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ~0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

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September 22, 2019

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

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September 22, 2019

High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant.

Salvia splendens Ker-Gawler, scarlet or tropical sage, is a tender herbaceous perennial widely introduced and seen in public gardens all over the world. With few molecular resources, breeding is still restricted to traditional phenotypic selection, and the genetic mechanisms underlying phenotypic variation remain unknown. Hence, a high-quality reference genome will be very valuable for marker-assisted breeding, genome editing, and molecular genetics.We generated 66 Gb and 37 Gb of raw DNA sequences, respectively, from whole-genome sequencing of a largely homozygous scarlet sage inbred line using Pacific Biosciences (PacBio) single-molecule real-time and Illumina HiSeq sequencing platforms. The PacBio de novo assembly yielded a final genome with a scaffold N50 size of 3.12 Mb and a total length of 808 Mb. The repetitive sequences identified accounted for 57.52% of the genome sequence, and ?54,008 protein-coding genes were predicted collectively with ab initio and homology-based gene prediction from the masked genome. The divergence time between S. splendens and Salvia miltiorrhiza was estimated at 28.21 million years ago (Mya). Moreover, 3,797 species-specific genes and 1,187 expanded gene families were identified for the scarlet sage genome.We provide the first genome sequence and gene annotation for the scarlet sage. The availability of these resources will be of great importance for further breeding strategies, genome editing, and comparative genomics among related species.

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September 22, 2019

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X).The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions.We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.

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September 22, 2019

Large-scale gene losses underlie the genome evolution of parasitic plant Cuscuta australis.

Dodders (Cuscuta spp., Convolvulaceae) are root- and leafless parasitic plants. The physiology, ecology, and evolution of these obligate parasites are poorly understood. A high-quality reference genome of Cuscuta australis was assembled. Our analyses reveal that Cuscuta experienced accelerated molecular evolution, and Cuscuta and the convolvulaceous morning glory (Ipomoea) shared a common whole-genome triplication event before their divergence. C. australis genome harbors 19,671 protein-coding genes, and importantly, 11.7% of the conserved orthologs in autotrophic plants are lost in C. australis. Many of these gene loss events likely result from its parasitic lifestyle and the massive changes of its body plan. Moreover, comparison of the gene expression patterns in Cuscuta prehaustoria/haustoria and various tissues of closely related autotrophic plants suggests that Cuscuta haustorium formation requires mostly genes normally involved in root development. The C. australis genome provides important resources for studying the evolution of parasitism, regressive evolution, and evo-devo in plant parasites.

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September 22, 2019

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs?=?210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi.

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September 22, 2019

Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae.

The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene blaNDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.

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September 22, 2019

A mosaic monoploid reference sequence for the highly complex genome of sugarcane.

Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.

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September 22, 2019

Molecular characterization of invasive meningococcal isolates in Burkina Faso as the relative importance of serogroups X and W increases, 2008-2012.

Neisseria meningitidis serogroup A disease in Burkina Faso has greatly decreased following introduction of a meningococcal A conjugate vaccine in 2010, yet other serogroups continue to pose a risk of life-threatening disease. Capsule switching among epidemic-associated serogroup A N. meningitidis strains could allow these lineages to persist despite vaccination. The introduction of new strains at the national or sub-national levels could affect the epidemiology of disease.Isolates collected from invasive meningococcal disease in Burkina Faso between 2008 and 2012 were characterized by serogrouping and molecular typing. Genome sequences from a subset of isolates were used to infer phylogenetic relationships.The ST-5 clonal complex (CC5) was identified only among serogroup A isolates, which were rare after 2010. CC181 and CC11 were the most common clonal complexes after 2010, having serogroup X and W isolates, respectively. Whole-genome phylogenetic analysis showed that the CC181 isolates collected during and after the epidemic of 2010 formed a single clade that was closely related to isolates collected in Niger during 2005 and Burkina Faso during 2007. Geographic population structure was identified among the CC181 isolates, where pairs of isolates collected from the same region of Burkina Faso within a single year had less phylogenetic diversity than the CC181 isolate collection as a whole. However, the reduction of phylogenetic diversity within a region did not extend across multiple years. Instead, CC181 isolates collected during the same year had lower than average diversity, even when collected from different regions, indicating geographic mixing of strains across years. The CC11 isolates were primarily collected during the epidemic of 2012, with sparse sampling during 2011. These isolates belong to a clade that includes previously described isolates collected in Burkina Faso, Mali, and Niger from 2011 to 2015. Similar to CC181, reduced phylogenetic diversity was observed among CC11 isolate pairs collected from the same regions during a single year.The population of disease-associated N. meningitidis strains within Burkina Faso was highly dynamic between 2008 and 2012, reflecting both vaccine-imposed selection against serogroup A strains and potentially complex clonal waves of serogroup X and serogroup W strains.

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